脐带间充质干细胞外泌体对急性皮肤创面愈合的初步研究

Role of umbilical cord mesenchymal stem cell-derived exosomes in acute skin wound healing

目的探讨脐带间充质干细胞(ucMSC)外泌体对急性皮肤创面愈合的影响。方法用超高速离心法提取ucMSC外泌体,经透射电镜观察、Western印迹检测外泌体表面标志物CD63和TSG101和粒径分析鉴定外泌体。体外培养3~5代人皮肤成纤维细胞(HSF),分别用含0(阴性对照组)、1、2μg/ml外泌体的高糖DMEM培养基孵育24 h(分别为1、2μg/ml组),CCK8法检测ucMSC外泌体对HSF增殖活力的影响。取24只8周龄雄性BALB/c小鼠构建全层皮肤损伤小鼠模型,按随机数字表等分为ucMSC外泌体组和磷酸盐缓冲液(PBS)组,距创缘约1 mm处等距皮下多点注射ucMSC外泌体悬液或PBS。术后第0、4、7、10、14天观察两组小鼠创面并计算创面残余面积百分比,术后第7、14天取创面组织后行HE和Masson染色,观察皮肤组织结构变化。术后第14天收集两组小鼠创面皮肤组织,实时定量PCR和Western印迹检测Ⅰ型胶原蛋白、纤连蛋白、血管内皮生长因子mRNA和蛋白的表达水平。统计分析采用单因素方差分析、LSD-t检验、双因素重复测量方差分析及非配对t检验。结果透射电镜下,ucMSC外泌体呈椭圆形,直径约100 nm;Western印迹检测显示,ucMSC外泌体表面蛋白CD63、TSG101表达阳性;粒径分析显示,96.2%ucMSC外泌体直径30~150 nm。CCK8法检测显示,外泌体1μg/ml组和2μg/ml组HSF相对活力(0.97±0.05、1.08±0.07)均显著高于阴性对照组(0.71±0.04),t值分别为2.00、7.05,均P<0.05,且2μg/ml组HSF相对活力显著高于1μg/ml组,t=5.09,P<0.05。术后第4、7、10、14天,ucMSC外泌体组创面残余面积百分比均显著低于PBS组(均P<0.05)。HE和Masson染色显示,与PBS组相比,ucMSC外泌体组小鼠创面新生皮肤组织中毛囊、腺体以及肉芽组织更丰富且有新生血管形成,胶原排列也更整齐。实时定量PCR和Western印迹实验显示,ucMSC外泌体组Ⅰ型胶原蛋白、纤连蛋白、血管内皮生长因子mRNA和蛋白的表达均显著高于PBS组(均P<0.01)。结论皮下注射ucMSC外泌体可以促进小鼠急性皮肤创面愈合,可能与ucMSC外泌体促进小鼠创面组织中细胞外基质和血管内皮生长因子的合成及促进HSF增殖有关。

Objective To investigate the role of umbilical cord mesenchymal stem cell-derived exosomes(ucMSC-exos)in acute skin wound healing in mice.Methods ucMSC-exos were extracted by ultracentrifugation,and identified by transmission electron microscopy,Western blot analysis of exosome surface markers CD63 and TSG101,and particle size analysis.Firstly,in vitro cultured third-to fifth-passage human skin fibroblasts(HSF)were incubated with high-glucose Dulbecco′s modified Eagle′s medium(DMEM)containing 0,1 and 2μg/ml exosome suspension for 24 hours(negative control group,1-and 2-μg/ml groups,respectively),and cell counting kit-8(CCK8)assay was performed to evaluate the effect of ucMSC-exos on the proliferative activity of HSF.Secondly,24 male BALB/c mice aged 8 weeks were selected to construct a mouse model of full-thickness skin wound,and then divided into ucMSC-exos group and phosphate-buffered saline(PBS)group by using a random number table to be subcutaneously injected with exosome suspension and PBS respectively at multiple equidistant sites located about 1 mm apart from the wound edge.On days 0,4,7,10 and 14 after operation,the wounds in mice were observed,and the percentage of residual wound area was calculated in the above two groups.On days 7 and 14 after operation,wound tissues were resected and subjected to hematoxylin and eosin(HE)and Masson staining to observe structural changes of skin tissues.On day 14 after operation,wound tissues were collected in the two groups,and real-time quantitative PCR(qRT-PCR)and Western blot analysis were performed to determine the mRNA and protein expression of typeⅠcollagen,fibronectin and vascular endothelial growth factor,respectively.Statistical analysis was carried out by using one-way analysis of variance,least significant difference-t test,two-way repeated measures analysis of variance and unpaired t-test.Results Under the transmission electron microscope,the ucMSC-exos were oval in shape with a diameter of about 100 nm;Western blot analysis showed positive expression of ucMSC-exos surface proteins CD63 and TSG101;particle size analysis showed that 96.2%of the ucMSC-exos had diameters of 30-150 nm.CCK8 assay showed that the relative proliferative activity of HSF was significantly higher in the 1-and 2-μg/ml groups(0.97±0.05,1.08±0.07,respectively)than in the negative control group(0.71±0.04;t=2.00,7.05,respectively,both P<0.05),and significantly higher in the 2-μg/ml group than in the 1-μg/ml group(t=5.09,P<0.05).On days 4,7,10 and 14 after operation,the percentage of residual wound area was significantly lower in the ucMSC-exos group than in the PBS group(all P<0.05).HE and Masson staining showed increased numbers of hair follicles,glands and granulation tissues,more neovascularization,and neater arrangement of collagens in neonatal skin tissues of the mice in the ucMSC-exos group compared with the PBS group.qRT-PCR and Western blot analysis showed significantly increased mRNA and protein expression of typeⅠcollagen,fibronectin and vascular endothelial growth factor in the ucMSC-exos group compared with the PBS group(all P<0.01).Conclusion Subcutaneous injections of ucMSC-exos can promote acute skin wound healing in mice,likely by promoting the synthesis of extracellular matrix and vascular endothelial growth factor in wound tissues of mice and proliferation of HSF.